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| United States Patent |
5,133,908
|
|
Stainmesse
, et al.
|
July 28, 1992
|
Process for the preparation of dispersible colloidal systems of a
substance in the form of nanoparticles
Abstract
The Process according to the invention comprises:
(1) the preparation of a liquid phase consisting essentially of a solution
of the substance in a solvent or in a mixture of solvents to which may be
added one or more surfactants,
(2) the preparation of a second liquid phase consisting essentially of a
non-solvent of a mixture of non-solvents for the substance and to which
may be added one or more surfactants, the non-solvent or the mixture of
non-solvents for the substance being miscible in all proportions with the
solvent or the mixture of solvents for the substance,
(3) the addition of one of the liquid phases prepared in (1) or (2) to the
other with moderate stirring so as to produce a colloidal suspension of
nanoparticles of the substance and,
(4) if desired, the removal of all or part of the solvent or the mixture of
solvents for the substance and of the non-solvent or the mixture of
non-solvents for the substance so as to produce a colloidal suspension of
nanoparticles of the desired concentration or to produce a powder of
nanoparticles. Said substance may be a protein.
Field of use: chemistry, biochemistry, pharmacy, medicine, cosmetics.
| Inventors:
|
Stainmesse; Serge (Choisy Le Roi, FR);
Fessi; Hatem (Paris, FR);
Devissaguet; Jean-Phillppe (Neuilly S/Seine, FR);
Puisieux; Francis (Maisons Alfort, FR);
Theis; Curt (Saint Louis, MO)
|
| Assignee:
|
Centre National de la Recherche Scientifique (CNRS) (Paris, FR)
|
| Appl. No.:
|
374246 |
| Filed:
|
June 30, 1989 |
Foreign Application Priority Data
| Dec 31, 1986[FR] | 86 18446 |
| Jun 30, 1988[FR] | 88 08871 |
| Current U.S. Class: |
264/4.1; 264/4.3; 424/469; 424/485; 424/486; 424/487; 427/213.36; 514/965; 977/DIG.1 |
| Intern'l Class: |
A61K 009/51; A61K 009/64; B01J 013/08 |
| Field of Search: |
264/4.6,4.1
427/213.36
424/469,485,486,487
514/965
|
References Cited
U.S. Patent Documents
| 3691090 | Sep., 1972 | Kitajima et al. | 424/487.
|
| 4492720 | Jan., 1985 | Mosier | 427/213.
|
| 4585651 | Apr., 1986 | Beck et al. | 514/932.
|
| 4741872 | May., 1988 | DeLuca et al. | 424/487.
|
| Foreign Patent Documents |
| 495261 | Mar., 1977 | AU.
| |
| 201942 | Mar., 1939 | CH.
| |
Primary Examiner: Lovering; Richard D.
Attorney, Agent or Firm: Young & Thompson
Parent Case Text
The present application is a continuation-in-part of Ser. No. 07/140,245,
filed Dec. 31, 1987, and now abandoned.
Claims
We claim:
1. A process for the preparation of dispersible colloidal systems of a
protein in the form of spherical particles of the matrix type and of a
size less than 500 nm (nanoparticles), comprising:
combining (1) a first liquid phase consisting essentially of a solution of
the protein in water or in an aqueous mixture, said first liquid phase
being at a temperature lower than the coagulation temperature of the
protein; and (2) a greater volume of a second liquid phase consisting
essentially of water or an aqueous mixture at a temperature higher than
the coagulation temperature of the protein, which may contain a
biologically active substance and to which may be added one or more
surfactants;
thereby substantially instantaneously to precipitate from said first liquid
phase and said second liquid phase particles of said protein to produce a
colloidal suspension of nanoparticles of the protein.
2. The process according to claim 1, wherein the protein is a serum
albumin.
3. The process according to claim 1, wherein the protein is an elastin.
4. The process according to claim 1, wherein said first liquid phase
further comprises a biologically active substance.
5. The process according to claim 4, wherein the combining step is
performed such that the biologically active substance is bound to the
nanoparticles of protein.
6. The process according to claim 1, further comprising removing at least
part of the water or aqueous mixture in said colloidal suspension, so as
to form a colloidal suspension of a desired concentration or to form a
powder of nanoparticles.
7. The process according to claim 6, wherein all of the water in the
colloidal suspension is removed by lyophilization.
8. The process according to claim 1, wherein the first liquid phase has a
temperature of from 0.degree. to 50.degree. C.
9. The process according to claim 1, wherein the second liquid phase has a
temperature of from 80.degree. to 100.degree. C.
10. The process according to claim 1, wherein the pH of the combined first
and second liquid phases is two to three units removed from the
isoelectric point of the protein.
11. The process according to claim 1, wherein the protein is present in the
first liquid phase in a concentration of 0.1 to 10%.
12. The process according to claim 11, wherein the protein concentration is
from 0.5 to 4%.
13. The process according to claim 1, wherein the ratio volumes of the
first liquid phase to the second liquid phase is at least 0.1.
14. The process according to claim 13, wherein the ratio is from 0.1 to
0.6.
15. The process according to claim 1, wherein the nanoparticles have a size
of about 150 to 300 nm.
Description
The subject of the present invention is a novel procedure for the
preparation of dispersible colloidal systems of a substance in the form of
spherical particles of the matrix type and of a size less than 500 nm
(nanoparticles).
Sub-microscopic particles of a diameter less than 500 nm are already known,
in particular from the patents BE-A-808 034, BE-A-839 748, BE-A-869 107
and FR-A-2 504 408. According to BE-A-808 034 and -839 748, the
sub-microscopic particles are formed by micellar polymerization of a
monomer such as a derivative of acrylic acid. Similarly, BE-A-869 107 and
FR-A-2 504 408 describe the preparation of biodegradable nanoparticles
obtained by the polymerization of an alkyl cyano-acrylate and containing a
biologically active substance. The methods resort to polymerization in
solution and are thus limited to using a limited number of polymers which
can be prepared, in particular, by vinyl addition and are not suitable for
naturally occurring or semi-synthetic polymers. Furthermore, it is
difficult to control the molecular weight of the polymer constituting the
nanoparticles and it is necessary, particularly when their biological use
is under consideration, to remove the residual monomers and oligomers and,
where necessary, the excess reagents involved in the polymerization
reaction (initiator and catalyst), as well as the surfactants if they are
used at high concentration or are not biocompatible. In actual fact, the
purification often proves to be onerous (ultracentrifugation, dialysis)
since the filtration of the nanoparticles is not always possible on
account of their size.
Methods employing emulsification-evaporation have also been described which
make use of preformed polymers and according to which an organic solution
of polymer, immiscible with water, is emulsified in a continuous aqueous
phase, then the solvent is evaporated in order to give rise to a
suspension of polymer insoluble in water. However, while the major
relevance of this method lies in its applicability to numerous polymers of
synthetic or semi-synthetic origin and thus in the possibility of
producing nanoparticles from well-defined polymers, the disadvantage
associated with it resides in the difficulty of preparing ultra-fine and
homogeneous emulsions for the production of nanoparticles smaller than 500
nm and homogeneous in size. Furthermore, the necessity of often having to
use surfactants in high proportions (20%) and which must be removed, and
the use of sophisticated equipment with high energy requirements
(sonicators, homogenizers, . . . ) constitute serious handicaps to their
industrial application.
The production of nanoparticles has also been suggested in the case of
proteins, in particular by heat denaturation of a water-in-oil emulsion of
a solution of a protein such as albumin (Kramer, P. A.: J. Pharm. Sci.,
63, 1646 (1974), or by the desolvation of a protein solution such as
gelatin by means of a mineral salt or ethanol (Marty et al., in Austr. J.
Pharm. Sci., 6, 65 (1978) or in Pharm. Acta. Helv. 53, No. 1 (1978)),
followed, in both cases, by hardening by means of an aldehyde. The main
disadvantage of the method of Kramer is the need to emulsify beforehand
the aqueous solution of the macromolecular starting material in a
continuous oily phase. Since this emulsion must be very fine, the use of
surfactants and the necessary equipment (sonicator, etc) is essential for
the production of nanoparticles of appropriate size. As for the method of
Marty, it requires the use of considerable amounts of mineral salts which
must be removed, as well as the excess of aldehyde and the sulfite or
metabisulfite used to neutralize the latter.
All of the methods described above are only applicable to certain classes
of molecules and necessarily involve operations which are expensive
(ultracentrifugation, sonication, etc) or hard to control (polymerization)
without ensuring that the size of the particles is acceptably homogeneous
or even that the particles are sufficiently small (less than 500 nm) such
as would assure their long-term stability in the form of a colloidal
suspension.
The invention proposes a new process for the preparation of nanoparticles
which is free from the above-mentioned disadvantages and which can be used
both for naturally occurring and synthetic polymeric substances and for
various organic substances (medicines, lipids, etc) or minerals (salts,
pigments, etc) as well as for mixtures of them.
The subject of the present invention is a process for the preparation of
dispersible colloidal systems of a substance in the form of spherical
particles of the matrix type and of a size less than 500 nm
(nanoparticles), comprising:
(1) the preparation of a liquid phase consisting essentially of a solution
of the substance in a solvent or in a mixture of solvents to which one or
more surfactants may be added,
(2) the preparation of a second liquid phase consisting essentially of a
non-solvent or a mixture of non-solvents of the substance to which one or
more surfactants may be added, the non-solvent or the mixture of
non-solvents for the substance being miscible in all proportions with the
solvent or mixture of solvents for the substance,
(3) the addition of one of the liquid phases prepared in (1) or (2) to the
other with moderate stirring so as to produce a colloidal suspension of
nanoparticles of the substance, and
(4) if desired, the removal of all or part of the solvent or the mixture of
solvents for the substance and of the non-solvent or the mixture of
non-solvents for the substance so as to give rise to a colloidal
suspension of nanoparticles of the desired concentration or to lead to a
powder of nanoparticles.
In step (3), the nanoparticles are formed practically instantaneously. The
solution becomes milky-white and shows the Tyndall effect characteristic
of colloidal suspensions. At this stage, it is preferable to add the
liquid phase prepared in step (1) to the liquid phase prepared in step
(2), particularly if the latter is aqueous, but the reverse order is
possible as the examples show.
The "substance" used according to the process of the invention may be
practically any substance sufficiently soluble in a given solvent.
The "substance" may be, in particular, a polymer, either a synthetic
polymer, for example poly (d,l) lactic acid (PLA), etc, a semi-synthetic
polymer such as for example cellulose butyrate acetate, ethylcellulose,
the phthalate of hydroxymethyl-propylcellulose (HPMCP), etc, or a
naturally occurring polymer, for example gelatin, gum arabic, etc.
Numerous other polymers can be used, for example: the aceto-phthalate of
polyvinyl, the aceto-phthalate of cellulose; maleic acid derivatives (for
example "Gantrez"); the copolymers of acrylic acid and acrylates and
acrylic acid polymers (for example Eudragit.RTM.); d or l or (d,l)
polylactic acid; the copolymers of lactic acid and glycolic acid,
polypeptides, glycol derivatives (derivatives of propiolactone,
butyrolactone, pivalolactone, .epsilon.-caprolactone, etc); the polymers
obtained from cyclic esters of hydroxybutyric acid, hydroxyisobutyric
acid, hydroxymethylvaleric acid, phenyllactic acid, hydroxyethylbutyric
acid; poly beta benzyl malate; the copolymers of malic acid and benzyl
malate; a polyvinylpyrrolidone-vinyl acetate cross-linked copolymer, alkyl
polycyanoacrylates; poly (ethylene-vinyl acetate); water-soluble polymers
(gelatin, gum arabic, methylcellulose, etc); oligomers (styrene allyl
alcohol), etc.
The "substance" may be a fatty substance, for example a mono-, di-, or
tri-glyceride of a fatty acid; a hydrogenated oil such as, for example,
hydrogenated castor oil, an oil solid at room temperature, such as for
example copra oil; a wax such as for example beeswax, an ester formed
between a fatty acid and polyethylene glycol (PEG), as for example the
distearate of PEG 6000, etc.
The "substance" may also be a biologically active substance, in particular
a medicamentous active ingredient or a precursor of a medicamentous active
ingredient or even a contrasting agent or a biological reagent. In such a
case, it may be of interest to prepare nanoparticles comprising both a
biologically active substance and a polymer as "substance" as will be seen
later.
The "substance" may also be a pigment, an ink, a lubricant, an agent for
treating surfaces, etc.
It is obvious that the process according to the invention can be applied
equally to one substance or to several.
In this respect, according to a variant of the process, a second substance
may be bound by absorption to the surface of nanoparticles already formed
in step (3) by simple addition to the colloidal suspension of the polymer,
if necessary after concentration. This second substance may be in
particular a biologically active substance.
The "solvent" or the mixture of solvents used is a liquid capable of
dissolving the substance (for example, the polymer and/or the biologically
active substance). Moreover, the solvent must be miscible with the
non-solvent for the substance used in the preparation. Thus, in most
cases, the solvent will be an organic solvent such that the liquid phase
(1) will constitute the organic phase whereas the liquid phase (2) will
constitute the aqueous phase, but it is possible to use either two organic
phases or two aqueous phases provided the conditions regarding solubility,
insolubility and miscibility are met. On the other hand, the solvent must
be sufficiently volatile for it to be removed if necessary. For example,
in the case in which the substance is a polymer (to which a biologically
active substance has or has not been added), the solvent may be chosen
from among a lower alcohol (methanol, ethanol, isopropanol, etc), a lower
ketone (acetone, methyl-ethyl-ketone, etc), a light hydrocarbon or a
mixture of light hydrocarbons (hexane, petroleum ether, etc), a
chlorinated light hydrocarbon (chloroform, methylene chloride,
trichloroethylene, etc) or other common light solvents such as
acetonitrile, dioxane etc.
The "non-solvent" or the mixture of non-solvents for the substance is a
liquid which does not dissolve the substance while being miscible with the
solvent used. Thus, for example, when the substance is a polymer such as
PLA, the solvent may be acetone and the non-solvent may be ethanol or
distilled water; if the substance is for example an acrylic polymer such
as Eudragit L100.RTM., the solvent may be an alkaline aqueous phase and
the non-solvent may be an acidic aqueous phase. It may be advantageous to
add a low proportion (lower than 20% by volume, for example about 10% by
volume) of non-solvent to the solvent in liquid phase (1) as this will
make it possible to produce smaller nanoparticles, in particular particles
less than 100 nm.
In order to produce a more stable suspension it is desirable to add one or
more surfactants (or emulsifying agents). The surfactants may be anionic
(for example sodium laurylsulfate), cationic (for example, quaternary
ammonium) or non-ionic (for example, monoesters of sorbitan which may or
may not contain a polyoxyethylene residue, ethers formed between fatty
alcohols and polyoxyethylene glycols, polyoxyethylene-polypropylene
glycol, etc).
However, the nanoparticles may be produced according to the invention
without surfactants and, moreover, the latter are not necessary if all of
the solvents and non-solvents are eliminated in step (4), for example by
lyophilization. In this way, lyophilized nanoparticles may be produced
which can be stored for long periods.
The proportion of surfactants in the colloidal suspension prepared in step
(3) to which they have been added may vary in particular from 0.1% to 10%
by weight, and preferably between 0.2 and 2% by weight.
In the case in which the substance is a polymer, the concentration of the
polymer in the solvent or the mixture of solvents may vary between 0.1 and
10% , and preferably between 0.2 and 2% by weight.
The ratio of the volumes of the solvents and non-solvents must be such as
to allow the precipitation of the polymer. As this ratio increases, the
size of the nanoparticles diminishes.
The need for moderate agitation of the preparation in step (3) is dependent
on the amount of substances utilized. It is not necessary for small
quantities.
The effect of temperature and pH on the process according to the invention
are so limited that it is usually not necessary to work under special
conditions. However, when the two phases (1) and (2) used are aqueous,
their respective pHs must be different in order for them to comply with
the conditions of being a solvent and a non-solvent.
Moreover, the presence of an electrolyte (for example sodium chloride) does
not appear to affect the production of nanoparticles. Thus, after the
formation of the nanoparticles in Example 1, a concentration of 25 mg/ml
sodium chloride does not lead to coalescence or precipitation of the
nanoparticles formed.
The particles produced according to the invention can be autoclaved if the
physical properties of the substance permit this.
The process for the preparation of nanoparticles according to the invention
offers the following advantages compared with known processes:
the production of nanoparticles smaller than 500 nm and in particular of
about 200 nm by means of a simple method not requiring a supply of energy;
in the case in which the substance comprises a polymer, the nanoparticles
are no longer produced by polymerization of a monomer but by
"nanoprecipitation" of a well-defined polymer;
the utilization of naturally occurring polymers as well as synthetic
polymers which are known to be innocuous and which have been used for
medical purposes for a very long time;
the utilization of polymers which are of the type which are biocompatible;
the possibility of using polymers which can dissolve in the organism once a
particular pH value is attained, thus ensuring that polymer particles do
not accumulate in the organism;
the possibility of using polymers which by their nature are bioresorbable,
the products of their degradation being completely innocuous;
the production of spherical particles exhibiting only a slight variation in
size.
The following examples illustrate the invention. The nanoparticles obtained
are visible in the transmission electron microscope
(.times.25,000-150,000) and, after negative staining with phosphotungstic
acid, appear as approximately round, non-contrasted particles.
EXAMPLE 1
Preparation of nanoparticles of polymers
On the one hand, 125 mg of poly (d,l) lactic acid (P.L.A.) are dissolved in
25 ml of acetone and, on the other hand, 125 mg of the mixed polymer
formed between ethylene oxide and propylene glycol (Pluronic F68.RTM. or
Poloxamer 188), a non-ionic surfactant, are dissolved in 50 ml of purified
water.
The acetone phase is added to the aqueous phase with magnetic stirring (100
r.p.m.). The mixture immediately becomes opalescent as a result of the
formation of nanoparticles of the polymer (P.L.A.). The mean diameter of
the nanoparticles measured in a diffractometer equipped with a laser beam
(Nanosizer.RTM. supplied by the Coultronics Company) immediately after
preparation is about 200 nm with a dispersion index of 0.5.
The acetone is removed under reduced pressure (water pump vacuum) and the
suspension is concentrated to the desired volume, for example 10 ml, under
the same conditions.
The concentrated suspension of nanoparticles is filtered through a glass
frit (pores 9-15 .mu.m) or through a membrane filter (pores 5 .mu.m) and
the diameter of the nanoparticles, measured again in the filtrate, remains
unchanged as does the dispersion index. Examination in the transmission
electron microscope reveals the nanoparticles of poly (d,l) lactic acid to
be regular spheres.
After prolonged standing (18 months) the appearance of the suspension of
nanoparticles remains unchanged and, in particular, neither irreversible
sedimentation nor variation in the size of the nanoparticles is observed.
EXAMPLE 2
Variant of Example 1
The process described in Example 1 is employed but the aqueous phase is
added to the acetone phase. The nanoparticles produced have the same
properties as those in Example 1.
EXAMPLE 3
Variant of Example 1
The process described in Example 1 is employed but the acetone phase is
added to the aqueous phase without stirring. The nanoparticles produced
have a mean diameter of 205 nm with a mean dispersion index of 1.
EXAMPLE 4
Variant of Example 1
The process described in Example 1 is employed but without the addition of
a surfactant to the aqueous phase. The nanoparticles produced have a mean
diameter of 207 nm with a means dispersion index of 1.3.
EXAMPLE 5
Preparation of nanoparticles containing indomethacin (lipophilic active
ingredient)
a) The process described in Example 1 is employed but 5 mg of indomethacin
are added to the acetone phase. The nanoparticles produced have a mean
diameter of 180 nm with a mean dispersion index of 1.5. After
ultracentrifugation and titration of the indomethacin in the phase used as
dispersion medium, the amount of active ingredient incorporated into the
nanoparticles amounts to 80% of the amount initially present.
b) Pharmacological assay
When administered by the oral route to the fasted rat (5 mg/kg of
indomethacin) the suspension of nanoparticles leads to a more rapid and
more complete digestive absorption of the indomethacin than that observed
after administration of the same dose of indomethacin in solution. After
repeated administration to the fasted rat (5 mg/kg of indomethacin on 3
successive days) the suspension of nanoparticles results in improved
digestive tolerance, as evidenced by the number of ulcerations and
hemorrhages, compared with that observed after administration of the same
dose of indomethacin in solution.
When administered by the intravenous route to the rat (5 mg/kg of
indomethacin) the suspension of nanoparticles gives rise to a
chronological profile of plasma concentrations of indomethacin which
demonstrates an increased extravascular distribution of the active
ingredient compared with that found after injection of indomethacin in
solution (increase of the volume of distribution of indomethacin by a
factor of approximately 2), followed by slower elimination (increase of
the biological half-life of indomethacin by a factor of approximately 2).
EXAMPLE 6
Preparation of nanoparticles containing doxorubicin (hydrophilic active
ingredient)
a) The process described in Example 1 is employed but 12.5 mg of
doxorubicin are added to the aqueous phase. The nanoparticles produced
have a mean diameter of 288 nm with a mean dispersion index of 2. After
ultracentrifugation and titration of the doxorubicin in the phase used as
dispersion medium, the amount of active ingredient incorporated into the
nanoparticles amounts to 50% of the quantity initially present.
b) Pharmacological assay
When administered to the rat at a dose of 10 mg/kg for three days, the
suspension of doxorubicin nanoparticles showed a marked improvement in
cardiactoxicity of the active ingredient compared with its administration
in solution, results comparable with those observed by COUVREUR et al. (J.
Pharm. Sci., 71, p. 790, (1982) with nanoparticles prepared by
polymerization of isobutyl cyanoacrylate.
EXAMPLE 7
Binding of an active ingredient (doxorubicin) to nanoparticles of polymer.
The process described in Example 1 is employed. Subsequently, 12.5 mg of
doxorubicin are added to the suspension of nanoparticles of P.L.A.
concentrated to a volume of 10 ml. After 72 h of contact the mean diameter
of the nanoparticles is about 220 nm with a mean dispersion index of 2.
After ultracentrifugation and titration of the doxorubicin in the phase
used as dispersion medium, the amount of active ingredient bound to the
nanoparticles amounts to 32% of the quantity initially present.
EXAMPLE 8
Addition of non-solvent to the solvent phase.
The process described in Example 1 is employed but the polymer is dissolved
in an acetone/water (90/10, v/v) mixture instead of pure acetone. The
presence of a low proportion of non-solvent for the polymer in its solvent
leads to nanoparticles, the mean diameter of which is about 90 nm with a
mean dispersion index of 1.5.
EXAMPLE 9
Use of two aqueous phases
a) On the one hand, 625 mg of an acrylic polymer (Eudragit L 100.RTM.) are
dissolved in 125 ml of purified water to which 3.45 ml of 0.1 N sodium
hydroxide are added.
On the other hand, 625 mg of the mixed polymer formed from ethylene oxide
and propylene glycol (Pluronic F68.RTM.) are dissolved in 250 ml of
purified water to which 0.85 ml of glacial acetic acid is added.
The basic polymer aqueous phase is added to the acidic aqueous phase with
magnetic stirring. The nanoparticles of Eudragit L100.RTM. form
immediately, turning the medium opalescent. After concentration of the
suspension under reduced pressure, the mean diameter of the nanoparticles
is about 130 nm with a mean dispersion index of 2.3.
b) Preparation of tablets. The suspension of nanoparticles of Eudragit
L100.RTM. obtained above was used to prepare a tablet coating formulation
by atomization. The coated tablets showed that they were gastro-resistant
for at least 2 h at acid pH (gastric medium of the US Pharmacopoeia) but
released their active ingredient at neutral pH (intestinal medium of the
US Pharmacopoeia).
EXAMPLE 10
Use of two polar organic phases
On the one hand, 125 mg of poly (d,l) lactic acid are dissolved in 25 ml of
tetrahydrofuran.
On the other hand, 125 mg of the mixed polymer formed between ethylene
oxide and propylene glycol (Pluronic F 68.RTM.) are dissolved in 50 ml of
absolute ethanol.
The polymer phase is added to the ethanolic phase with magnetic stirring.
The nanoparticles of poly (d,l) lactic acid form immediately, turning the
medium opalscent. After concentration of the suspension to a volume of 4
ml under reduced pressure and at low temperature, followed by filtration
through a glass frit (pores 9-15 .mu.m), the mean diameter of the
nanoparticles is about 201 nm with a mean dispersion index of 1.6.
EXAMPLE 11
Use of two apolar organic phases.
a) On the one hand, 125 mg of acrylic polymer (Eudragit L100.RTM.) are
dissolved in 25 ml of chloroform.
On the other hand, 0.2 ml of sorbitan mono-oleate (SPAN 80.RTM., non-ionic
surfactant) are dissolved in 50 ml of heptane.
The chloroform phase is added to the heptane phase with magnetic stirring.
The nanoparticles of Eudragit L100.RTM. form immediately, turning the
medium opalescent.
After concentration of the suspension to a volume of 30 ml, the
nanoparticles have a mean diameter of 350 nm with a mean dispersion index
of 1.
b) Preparation of tablets. When used under the same conditions as those in
Example 9b, the suspension of nanoparticles of Eudragit L100.RTM. enabled
gastro-resistant, coated tablets to be manufactured.
EXAMPLE 12
Preparation of nanoparticles of lipid
On the one hand, 125 mg of the stearate of glycerol are dissolved in an
acetone/tetrahydrofuran (90/10, v/v) mixture.
On the other hand, 0.25 ml of the polyoxyethylene derivative of sorbitan
mono-oleate (TWEEN 80.RTM.), non-ionic surfactant) are dissolved in 50 ml
of purified water.
The lipid organic phase is added to the aqueous phase with magnetic
stirring. The nanoparticles of the stearate of glycerol form immediately,
turning the medium opalescent.
After concentration of the suspension to a volume of 10 ml, the
nanoparticles have a mean diameter of 300 nm with a mean dispersion index
of 3.
EXAMPLE 13
Variant of Example 12
On the one hand, 125 mg of the palmito-stearate of glycerol and 0.1 ml of
sorbitan mono-oleate (SPAN 80.RTM.) are dissolved in 50 ml of absolute
ethanol.
On the other hand, 0.1 ml of the polyoxyethylene derivative of sorbitan
mono-oleate (TWEEN 80.RTM.) are dissolved in 50 ml of purified water.
The lipid alcoholic phase is added to the aqueous phase with magnetic
stirring. The nanoparticle of the palmito-stearate of glycerol form
immediately, turning the medium opalescent.
After concentration of the suspension to a volume of 10 ml, the
nanoparticles have a mean diameter of 160 nm with a mean dispersion index
of 2.
EXAMPLE 14
Preparation of nanoparticles of indomethacin
a) The process described in Example 1 is employed but the polymer is
replaced by 25 mg of indomethacin.
After concentration of the suspension by evaporation of the organic
solvent, the nanoparticles of indomethacin of a mean diameter of 290 nm
with a mean dispersion index of 2. Examination in the transmission
electron microscope reveals that the nanoparticles of indomethacin are
spherical and non-crystalline.
b) Pharmacological assay
When administered by the oral route to the fasted rat (5 mg/kg), the
suspension of nanoparticles of indomethacin leads to more rapid and more
complete absorption than that observed after administration of equivalent
doses of indomethacin in solution.
When administered by the intravenous route to the rat (5 mg/kg), the
suspension of nanoparticles of indomethacin gives rise to a chronological
profile of plasma concentrations of indomethacin which demonstrates an
increased extravascular distribution of the active ingredient compared
with that found after injection of equivalent doses of indomethacin in
solution (increase of the volume of distribution), followed by slower
elimination (increase of the biological half-life).
EXAMPLE 15
Lyophilization of nanoparticles of polymer.
The process described in Example 1 is employed. After concentration of the
suspension of nanoparticles of P.L.A. to a volume of 20 ml, 200 mg of
trehalose are added and the suspension is lyophilized.
After dispersion of the lyophilisate in 10 ml of purified water, the
nanoparticles have a mean diameter of 275 nm with a mean dispersion index
of 1.5.
EXAMPLE 16
Stability of nanoparticles of polymer at varying ionic strengths.
The process described in Example 1 is employed. After concentration of the
suspension of nanoparticles of P.L.A. to a volume of 10 ml, increasing
amounts of sodium chloride are progressively added to it. The suspension
of nanoparticles is perfectly stable when the concentration of sodium
chloride renders it isotonic with blood and remains so up to
concentrations of sodium chloride more than three times higher than the
isotonic concentration.
EXAMPLE 17
Preparation of nanoparticles in the presence of a salt.
The process described in Example 1 is employed but 90 mg of sodium chloride
is added to the aqueous phase. After concentration of the suspension of
nanoparticles to a volume of 10 ml, corresponding to a sodium chloride
concentration with the isotonicity of blood, the nanoparticles have a mean
diameter of 250 nm with a mean dispersion index of 2.
The suspension remains stable with time and, after being stored for 12
months, shows neither irreversible sedimentation nor variation in the size
of the nanoparticles.
It is possible that the nanoparticles produced according to the invention
will find uses in many areas of technology.
As "vectors" of medicines in human and animal therapy, the nanoparticles
offer the prospects of:
attaining new sites of action, in particular intracellular sites, and even
intralysosomal sites;
using new routes of administration for known medicines by increasing the
stability and/or the absorption of the medicines, or by making available
insoluble medicines in forms which can be injected by the intravascular
route;
modifying the tissue distribution of the medicines by better targetting
towards favourable sites of action and/or by diverting them from sites at
which they produce undesirable, or even toxic, effects (improvement of the
therapeutic index).
In pharmacy, the colloidal dispersions in the form of nanoparticles make it
possible to:
prepare injectable forms of insoluble medicines,
stabilize a medicamentous active ingredient, and
prepare coatings of galenical forms starting from aqueous dispersions of
film-forming polymers.
In the field of agrochemistry, the nanoparticles can be used as vehicles
for insecticides, pesticides, etc . . . Their size leads to the
expectation of a more powerful action as a result of better penetration
through the cuticle. The low viscosity of the dispersion enables
atomization to be carried out very easily in the form of droplets of very
small size which are more efficacious because they provide more intimate
covering.
In the area of paints, varnishes and treatment of surfaces in general, the
nanoparticles may function as vehicles of pigments, reagents, strippers,
etc . . . in the form of aqueous dispersions of very low viscosity, easy
to atomize or apply and which can, if necessary, be made viscous and even
adhesive (resuspension of the nanoparticles in an appropriate vehicle). A
small size of the nanoparticles leads to a very fine deposition and to a
very high homogeneity, for example, of pigmentation.
The nanoparticles produced according to the invention can also be used in
the fields of printing and reproduction graphics, the treatment of
surfaces of textiles and fibres, photography, lubrication, etc . . .
According to a further embodiment, the said substance is a protein and
optionally a biologically active substance, the said solvent is water or
an aqueous mixture at a temperature lower than the coagulation temperature
of the protein, the said non-solvent for the substance is water at a
temperature higher than the coagulation temperature of the protein and may
optionally contain a biologically active substance, and the said two
liquid phases (1) and (2) are added to each other under conditions of pH
far removed from the isoelectric point of said protein.
The object of the present invention is also process for the preparation of
dispersible colloidal systems of a protein in the form of spherical
particles of the matrix type and of a size less than 500 nm
(nanoparticles).
The present process comprises:
(1) the preparation of a liquid phase consisting essentially of a solution
of the protein and optionally of a biologically active substance in water
or in an aqueous mixture at a temperature lower than the coagulation
temperature of the protein, and to which may be added one or more
surfactants,
(2) the preparation of a second liquid phase consisting essentially of
water or an aqueous mixture at a temperature higher than the coagulation
temperature of the protein, which may contain a biologically active
substance and to which may be added one or more surfactants,
(3) the addition with gentle stirring of one of the liquid phases obtained
in (1) or (2) to the other under conditions of pH far removed from the
isoelectric point of the protein so as to form almost instantaneously a
colloidal suspension of nanoparticles of the protein and optionally of the
biologically active substance, and
(4) if desired, the removal of all or part of the water or aqueous mixture
so as to form a colloidal suspension of nanoparticles of the desired
concentration or to form a powder of nanoparticles.
The protein is in particular a naturally occurring protein such as serum
albumin (for example human or bovine serum albumin) or elastin (bovine,
etc). The biologically active substance may be a medicamentous active
principle or a medicamentous precursor, a biological reagent or a cosmetic
principle. The invention makes it possible to obtain nanoparticles of
protein alone (which can be used as such) or in association with the
biologically active substance. It is also possible and even desirable in
the case of thermalinstability to bind the biologically active substance
to the protein nanoparticles already formed.
The solvent of phase (1) which is water or an aqueous mixture (for example
acidified or basified water) has in particular a temperature from
0.degree. to 50.degree. C., for example about room temperature.
The non-solvent for the protein in phase (2), which is water or an aqueous
mixture (for example acidified or basified water) has in particular a
temperature from 80.degree. to 100.degree. C. (at atmospheric pressure),
for example about boiling point.
The pH of the mixture of phases (1) and (2) must be far removed from the
isoelectric point of the protein in order to avoid its flocculation. This
desirable difference of pH is of the order of 2 to 3 units. Since the
naturally occurring proteins often have a pH of the order of 5 to 6, it is
desirable that the final solution has a pH of about 3 or about 9. For this
purpose, either acid or base may be added as appropriate to phase (1) or
(2). By "gentle stirring", a stirring up to 500 rpm, e.g. about 100 rpm is
meant, such as magnetic stirring.
The protein concentration in phase (1) may be from 0.1 to 10%, and
preferably from 0.5 to 4%.
The ratio of the volumes of phase (1)/phase (2) may be from 0.1 to 1, and
preferably from 0.2 to 0.6.
Finally, the colloidal solution of nanoparticles can be concentrated,
sterilized, buffered (for example at physiological pH), lyophilized or
cross-linked, as desired.
The invention makes it possible to obtain protein nanoparticles of, in
particular, from 150 to 300 nm.
The following examples illustrate the invention:
EXAMPLE 1
Preparation of nanoparticles of human serum albumin (HSA)
______________________________________
Phase 1
HSA 1.0 g
N hydrochloric acid 0.3 g
demineralized or distilled water
100.0 g
at room temperature
Phase 2
demineralized or distilled water
180.0 g
heated to boiling
______________________________________
Phase 1 is added to phase 2 with magnetic stirring. The mixture immediately
becomes opalescent as the result of the formation of HSA nanoparticles.
The mean size of the nanoparticles, measured in laser beam diffractometer
(Nanosizer.RTM. from Coultronics) is 190 nm with a mean dispersion index
of 0.5.
The suspension may be concentrated under reduced pressure to the desired
volume, for example 100 cm.sup.3.
EXAMPLE 2
Preparation of sterile human serum albumin nanoparticles
The procedure is the same as in example 1, then the suspension is
sterilized in an autoclave at 134.degree. C. for 15 minutes. The mean size
of the particles remains practically unchanged after sterilization.
EXAMPLE 3
Preparation of lyophilized human serum albumin nanoparticles.
The procedure is the same as in example 2, then the sterile suspension is
lyophilized.
The addition of a cryoprotector (maltose, trehalose . . . etc) 1 is not
essential, but promotes the resuspension of the lyophilisate. The mean
size of the particles remains unchanged after lyophilization.
EXAMPLE 4
Preparation of cross-linked human serum albumin nanoparticles.
The procedure is the same as in example 1, except that 0.06 g of a 25%
(wt/v) aqueous solution of glutaraldehyde is added to phase 1. The mean
size of the particles remain unchanged after cross-linking.
EXAMPLE 5
Preparation of bovine serum albumin nanoparticles (BSA).
The procedure is the same as in example 1, except that the HSA is replaced
by BSA and normal hydrochloric acid is replaced by the same amount of
0.01N sodium hydroxide. The mean size of the nanoparticles is 150 nm with
a mean dispersion index of 0.5.
The BSA nanoparticles may be cross-linked, sterilized in an autoclave and
lyophilized just like those of HSA.
EXAMPLE 6
Preparation of elastin nanoparticles.
The procedure is the same as in example 1 except that the HSA is replaced
by elastin. The mean size of the nanoparticles is 280 nm with a mean
dispersion index of 1.
The elastin nanoparticles may be cross-linked, sterilized in an autoclave
and lyophilized just like those of HSA.
EXAMPLE 7
Adsorption of an active principle on protein nanoparticles.
Increasing amounts (from 0.25 g to 2.50 g) of sodium salicylate are added
to nanoparticles prepared according to example 1 (HSA) or according to
example 5 (BSA). The level of binding of the sodium salicylate to the
nanoparticles, measured after ultracentrifugation, is 60% of the amount
used, irrespective of the amount of active principle added.
EXAMPLE 8
Preparation of nanoparticles in the presence of an active principle.
The procedure uses as in example 1 (1 g of HSA) or of example 5 (1 g of
BSA), but carried out in the presence of 0.50 g of sodium salicylate
dissolved in phase 1. The mean size of the nanoparticles is 200 nm with a
mean dispersion index of 0.5. The level of binding of the sodium
salicylate to the nanoparticles, measured after ultracentrifugation, is
60% of the amount used.
EXAMPLE 9
Variant of example 8.
The procedure is that of example 8, except that sodium salicylate is
dissolved in phase 2. The nanoparticles obtained have the same properties
as those of example 8.
EXAMPLE 10
Preparation of protein nanoparticles of doxorubicin.
50 mg of doxorubicin hydrochloride are added to nanoparticles prepared
according to example 1 (HSA) or according to example 5 (BSA). The level of
binding of doxorubicin to the nanoparticles, measured after
ultracentrifugation, is 90% of the quantity used.
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